Embolic agent with open type porous structure for slowly releasing tripterygium wilfordii

Abstract

The invention relates to the technical field of medical treatment, in particular to an embolic agent for slowly releasing triptolide with an open type porous structure. The embolization agent is triptolide-loaded perforated PBVHx microspheres, and the microspheres are marked as TP (at) OPMs; the TP (at) OPMs has a porous structure of which the surface is provided with pores and the internal pores are mutually communicated, PBVHx contains 90 mol% of 3-hydroxybutyric acid, 3 mol% of 3-hydroxyvaleric acid and 7 mol% of 3-hydroxyhexanoic acid, and the molecular weight is 46 kDa. According to the embolic agent for slowly releasing the triptolide with the open type porous structure provided by the invention, a novel non-lactic acid polymer PBVHx is used as a basis, and an improved double emulsion method (G / O / W) is adopted to prepare a novel triptolide (TP)-loaded open pore microsphere (TP-coated OPMs) which is used for chemoembolization treatment.

Description

Technical Field

[0001] This invention relates to the field of medical technology, and more particularly to an embolic agent containing sustained-release tripterygium wilfordii with an open porous structure. Background Technology

[0002] Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide, attracting significant attention due to its high incidence and mortality rates, making it a key research focus in the fields of human health and public health. Surgical resection of the tumor is the primary strategy for cancer treatment, but postoperative complications such as tumor recurrence, metastasis, and bleeding can lead to poor prognosis or even treatment failure. Chemotherapy remains one of the preferred treatment options for HCC, but its severe side effects limit its clinical efficacy. Therefore, there is an urgent need to develop novel, highly effective, and safe anti-hepatocellular carcinoma drugs and establish optimized transcatheter arterial chemoembolization (TACE) systems. These advances are crucial for the clinical treatment of unresectable HCC and can bring significant survival benefits to patients.

[0003] With the deepening research into biocompatible materials and oncology, innovative and effective drug delivery technologies are being widely explored to improve cancer treatment outcomes. Currently, an increasing number of active ingredients from traditional Chinese medicine are receiving research attention. Tripterygium wilfordii (TP), a diterpenoid compound extracted from Tripterygium wilfordii, has attracted considerable attention due to its broad-spectrum antitumor properties. Its mechanisms of action include inhibiting the TNF / NF-κB / BCL2 signaling pathway, p53-mediated apoptosis, and regulating the c-Myc / miRNA cluster / target gene axis. Compared with traditional anti-hepatocellular carcinoma drugs (such as doxorubicin, sorafenib, and cisplatin), TP shows significantly greater efficacy against hepatocellular carcinoma. However, the potential hepatotoxicity of TP severely limits its clinical application, and as a hydrophobic drug, TP exhibits poor pharmacokinetic properties. To address this issue, encapsulating TP in hydrophobic biopolyesters or hydrogels to achieve controlled drug release can reduce the dosage of TP, improve antitumor efficacy, and thus become a key strategy for mitigating side effects. Hydrogels possess excellent flowability and biodegradability, and are often used as drug-releasing embolic agents to reduce the toxicity of hydrophilic drugs and maintain sustained anticancer effects. However, TP, as a typical hydrophobic drug, cannot be stably dispersed in hydrogels.

[0004] In recent years, biodegradable drug-loaded embolization microspheres prepared from polylactic acid (PLA) and its derivatives (such as polylactic acid-glycolic acid copolymer, PLGA) have become a research hotspot in the field of drug delivery and embolization therapy. Zeng et al. optimized formulations based on 20 different compositions to prepare PLGA microspheres with an average particle size of 42.36 μm, a drug content of 7.96%, a drug encapsulation efficiency of 80.16%, and an initial release rate of 14.48%. These microspheres achieved sustained TP release for 4 weeks in vitro. Similarly, Wu et al. prepared PLGA nanoparticles functionalized with methoxy polyethylene glycol-5-S-hexadecyl (mPEG-SSC) and soybean lecithin for co-delivering doxorubicin (DOX) and TP. Through self-assembly, these hydrophobic compounds were successfully loaded into PLGA nanoparticles, showing significant synergistic effects in both in vitro and in vivo studies. Furthermore, TP promoted the uptake of DOX by KB cells (human oral squamous cell carcinoma cell line). Li et al. prepared PLGA nanoparticles (TPL@mPLGA) that mimicked tumor cell membranes for loading TP to treat hepatocellular carcinoma. These nanoparticles had a particle size of 195.5±7.5 nm, a zeta potential of -21.5±0.2 mV, and a drug loading of 2.94%. They exhibited good stability and reduced TP toxicity while enhancing antitumor efficacy, providing a promising approach for hepatocellular carcinoma treatment. While PLGA or PLA microspheres are widely used in tissue engineering and drug delivery systems, the lactic acid released from these polymers may pose potential risks to tissues. Lactic acid accumulation can lead to acidification of the tumor microenvironment, potentially promoting tumor invasion and metastasis. Furthermore, lactic acid accumulation may suppress immune responses, allowing tumors to evade immune surveillance and further driving cancer progression.

[0005] Polyhydroxyalkanoates (PHAs) are a class of biopolymers synthesized by microorganisms, possessing biodegradability and widely used in tissue engineering and drug delivery. Unlike the strongly acidic lactic acid (pK=3.8) released by PLGA and PLA, PHA releases 3-hydroxybutyric acid (3HB), which is milder (pK=4.5). Furthermore, at similar molecular weights, the enzyme-dependent degradation rate of PHA is approximately three times that of PLA or PLGA, making it more advantageous for controlled drug release. In 1999, the first-generation PHA material, polyhydroxybutyric acid (PHB), was used as a potential chemoembolization agent. Alregib et al. prepared microspheres using the second-generation PHA material, polyhydroxybutyric acid-3-hydroxyvalerate copolymer (PHBV), and simultaneously loaded the radiopharmaceutical samarium-153 (153Sm) and the chemotherapeutic drug doxorubicin (Dox), forming Dox-153Sm-PHBV microspheres for chemoembolization therapy in advanced liver cancer. Zhang et al. used the third-generation PHA material poly(3-hydroxybutyric acid-co-3-hydroxyhexanoic acid) (PHBHHx) to design microspheres with a particle size comparable to capillaries (5-10 μm), for green fluorescent protein gene delivery in pancreatic vascular embolization. In addition to embolization applications, nanoscale PHA particles, due to their excellent biocompatibility and controllable degradation properties, have also been used in the treatment of various cancers.

[0006] Poly(3-hydroxybutyric acid-co-3-hydroxyvalerate-co-3-hydroxyhexanoic acid) (PBVHx, also known as PHBVHHx) is the newest member (fifth generation) of the PHA family, composed of 3-hydroxybutyric acid, 3-hydroxyvalerate, and 3-hydroxyhexanoic acid. Compared to other PHA members, this material is easier to process into injectable microspheres, exhibits superior biocompatibility and tunable biodegradability, and has been successfully processed into various microsphere and nanoparticle morphologies for tissue repair and drug delivery. In addition to traditional hermetically sealed or hollow microspheres, Wei et al. found that the structure of PBVHx microspheres facilitates the migration of surrounding cells to the microsphere core, which may improve embolization efficacy.

[0007] Therefore, this invention proposes an embolizing agent with an open porous structure of sustained-release triptolide. Using a modified double emulsion method (G / O / W), open-pore PBVHx microspheres loaded with triptolide (TP) were prepared as a novel chemoembolizing agent for human use. Summary of the Invention

[0008] Therefore, it is necessary to provide an embolic agent with an open porous structure for sustained-release tripterygium wilfordii to address the aforementioned technical problems.

[0009] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: The embolizing agent is a pore-filled PBVHx microsphere loaded with triptolide, denoted as TP@OPMs; The TP@OPMs have a porous structure with surface openings and interconnected internal pores.

[0010] As a preferred embodiment of the embolic agent of sustained-release tripterygium wilfordii with an open porous structure provided by the present invention, the PBVHx contains 90 mol% of 3-hydroxybutyric acid, 3 mol% of 3-hydroxyvalerate and 7 mol% of 3-hydroxyhexanoic acid, with a molecular weight of 46 kDa.

[0011] A preparation method for preparing the TP@OPMs, comprising the following steps: A. Dissolve PBVHx and TP together in dichloromethane; B. Mix the ammonium bicarbonate aqueous solution with the solution obtained in step A, and then homogenize and emulsify to form a G / O primary emulsion; C. Pour the G / O pre-emulsion into a polyvinyl alcohol aqueous solution, stir to evaporate dichloromethane, and form a G / O / W double emulsion; D. After centrifugation, washing, and freeze-drying, TP@OPMs were obtained.

[0012] In a preferred embodiment of the preparation method provided by the present invention, the mass ratio of PBVHx to TP is 25:1.

[0013] In a preferred embodiment of the preparation method provided by the present invention, the ammonium bicarbonate aqueous solution has a mass-volume concentration of 20%.

[0014] In a preferred embodiment of the preparation method provided by the present invention, the mass-volume concentration of the polyvinyl alcohol aqueous solution is 1%.

[0015] The application of the TP@OPMs in the preparation of chemoembolization agents for the treatment of hepatocellular carcinoma.

[0016] Application of the TP@OPMs in the preparation of vascular embolization devices for inducing ischemic necrosis of tumors.

[0017] It is clear without a doubt that the technical solution described above in this application can solve the technical problem that this application aims to address.

[0018] Meanwhile, through the above technical solutions, the present invention has at least the following beneficial effects: 1. The present invention provides an embolizing agent for sustained-release triptolide with an open porous structure. Based on the novel non-lactic acid polymer PBVHx, a novel open-pore microsphere (TP@OPMs) loaded with triptolide (TP) was successfully prepared by using a modified double emulsion method (G / O / W) for chemoembolization therapy.

[0019] 2. The TP@OPMs of this invention have an open-pore structure, exhibiting excellent sustained release performance of TP, with a release time far exceeding 21 days. In addition, the anti-tumor effect of TP@OPMs on hepatocellular carcinoma (HCC) cells is superior to that of TP-loaded sealed microspheres (TP@SMs). In in vivo central artery embolization experiments, TP@OPMs showed the best embolization effect, inducing progressive ischemic necrosis and tissue sloughing. Therefore, TP@OPMs are not only an effective drug delivery system, but also have significant potential in embolization applications, providing a promising strategy for the treatment of hepatocellular carcinoma and other malignant tumors. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a schematic diagram of the TP@OPMs of the present invention and the control group pSMs, pOPMs and TP@SMs; Figure 2 This is a schematic diagram of the structure of TP@OPMs of the present invention; Figure 3 This is a schematic diagram of the in vitro TP release and degradation of the PBVHx microspheres of the present invention; Figure 4 This is a schematic diagram illustrating the evaluation of PBVHx microspheres as a potential in vivo embolizing agent according to the present invention; Figure 5 This is a schematic diagram illustrating the in vitro antitumor activity of pSMs, pOPMs, TP@OPMs, and TP@SMs against HepG2 cells in accordance with the present invention. Figure 6 This is a schematic diagram of hemolysis of TP@OPMs of the present invention and control groups pSMs, OPMs, and TP@SMs; Figure 7 This is a schematic diagram illustrating the embolic effects of TP@OPMs, pSMs, pOPMs, TP@SMs, and control glycerol and commercially available gelatin particles (cGPs) of the present invention. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0023] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described.

[0024] It should be noted that, unless otherwise specified, the embodiments and features and technical solutions in the embodiments of the present invention can be combined with each other.

[0025] Example 1 An embolic agent containing sustained-release tripterygium wilfordii with an open porous structure.

[0026] 1. Materials and Methods 1.1 Materials Poly(3-hydroxybutyric acid-co-3-hydroxyvalerate-co-3-hydroxyhexanoic acid) (PBVHx, also known as PHBVHHx), containing 90 mol% 3-hydroxybutyric acid (3HB), 3 mol% 3-hydroxyvalerate (3HV), and 7 mol% 3-hydroxyhexanoic acid (3HHx) (molecular weight = 46 kDa), was provided by Blue Crystal Microbiology (China). Tripterygium wilfordii (TP), ammonium bicarbonate (AB), and polyvinyl alcohol (PVA) 1788 were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Lipase and glutaraldehyde solution were purchased from Maclean Reagent Co., Ltd. (Shanghai, China). Cell culture-related reagents, including endothelial cell culture medium (ECM), fetal bovine serum (FBS), and penicillin / streptomycin, were purchased from Science Cell Research Laboratories, Inc., USA. Cell counting kit-8 (CCK-8) was purchased from Dojin Chemical Research Institute (Kumamoto, Japan). Calcein-AM and 3,8-diamino-5-[3-(diethylmethylammonium)propyl]-6-phenylphenanthridine diiodide (PI) were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). All other chemical reagents used were of analytical grade.

[0027] 1.2 Preparation of TP-loaded PBVHx embolic microspheres Porous PBVHx microspheres (TP@OPMs) loaded with TP were prepared using an oil-in-gas-in-water (G / O / W) double emulsion method. The specific steps are as follows: Dissolve 250 mg PBVHx powder and 10 mg TP powder in dichloromethane (DCM) to prepare a 2.5% (w / v) solution. Dissolve ammonium bicarbonate in distilled water to prepare a 20% solution by mass / volume.

[0028] AB solution and PBVHx solution are mixed in a specific volume ratio and homogenized for a specific time to form G / O promulgation. The resulting emulsion was poured into a 1% PVA solution and magnetically stirred for 12 hours to ensure complete evaporation of dichloromethane.

[0029] TP-loaded sealed PBVHx microspheres (TP@SMs) were prepared by an oil-in-water (O / W) single emulsion evaporation method: 250 mg of PBVHx powder and 10 mg of TP were dissolved in dichloromethane, and the resulting solution was poured into a 1% PVA solution. The mixture was stirred at 500 rpm for 12 hours on a magnetic stirrer to allow the dichloromethane to evaporate completely.

[0030] Unloaded TP-free pure open-pore PBVHx microspheres (pOPMs) and pure sealed PBVHx microspheres (pSMs) were prepared. All microspheres were collected by centrifugation, washed three times with distilled water, freeze-dried, and stored in a desiccator for later use.

[0031] 1.3 Characterization of PBVHx embolic microspheres The surface morphology of PBVHx embolization microspheres was observed using a scanning electron microscope (SEM, Zeiss Sigma 300, Germany). The SEM images were analyzed using ImageJ 1.40 G software (National Institutes of Health, USA) to determine the average particle size and particle size distribution of the microspheres. The microspheres were cut using the WEI frozen ordered sectioning method (WEI FOSM) to observe their cross-sectional structure.

[0032] To verify the successful loading of the drug onto PBVHx microspheres, Fourier transform infrared (FTIR) spectroscopy was performed to compare unloaded and drug-loaded PBVHx microspheres. The original TP and TP-loaded PBVHx microspheres were mixed with potassium bromide (KBr) and compressed into tablets. Spectroscopy was performed using a Nicolet iS20 spectrometer (Thermo Fisher Scientific) at 500-4000 cm⁻¹. -1 Record FTIR spectra within the wavenumber range.

[0033] 1.4 Encapsulation efficiency of TP Encapsulation efficiency (EE) of TP in PBVHx microspheres with different structures was determined: TP was scanned across the entire wavelength range of 200-800 nm to determine the maximum absorption wavelength; TP solutions with concentrations of 0.01, 0.02, 0.03, 0.04, and 0.05 mg / mL were prepared in ethanol, and their absorbance was measured and a standard curve was plotted.

[0034] Dissolve TP@OPMs and TP@SMs separately in dichloromethane and stir continuously. Add an equal volume of ethanol to the dichloromethane solution, vortex for 5 minutes, and centrifuge for 15 minutes. Transfer the supernatant and repeat this operation 4 times. Combine the ethanol extracts and determine the TP concentration using ultraviolet spectrophotometry. Calculate the encapsulation efficiency using the following formula:

[0035] In the formula, Wa is the total mass of TP, and Wb is the mass of unencapsulated TP in the aqueous phase.

[0036] 1.5 In vitro release of TP from PBVHx embolic microspheres Disperse 10 mg of TP@SMs or TP@OPMs in 5 mL of phosphate-buffered saline (PBS, pH 7.4) and place the solution into a dialysis bag (molecular weight cutoff = 3.5 kDa, JELAP, China). Immerse the dialysis bag in 35 mL of PBS in a centrifuge tube and incubate at 37°C and 150 rpm. Collect 2 mL of release medium at specific time intervals and replenish with fresh PBS. Mix the extracted PBS with ethanol at a ratio of 1:10 and determine the TP concentration using a UV spectrophotometer. Repeat each experiment three times and calculate the cumulative TP release.

[0037] 1.6 In vitro cell compatibility of pure PBVHx microspheres without TP loading The cell compatibility of unloaded pure PBVHx microsphere extract with human vascular smooth muscle cells (HVSMCs, purchased from Shanghai Cell Bank, Chinese Academy of Sciences) was evaluated using the Cell Counting Kit-8 (CCK-8) and the live / dead cell viability assay kit (Ingenie Biotech, Inc., USA).

[0038] The cytotoxicity of microspheres was assessed using an indirect contact method: sterilized pSMs and pOPMs were dispersed in ECM containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin to prepare a dispersion with a concentration of 10 mg / mL; the dispersion was incubated at 37°C and 5% CO2 for 24 hours; the dispersion was filtered through a 0.22 μm filter membrane to obtain the extract, and its cytotoxicity was assessed according to ISO 10993-5:511.

[0039] HVSMCs are divided into 5×10 3Cells were seeded at a density of 1 cell / well in 96-well plates and incubated adherently for 24 hours. The medium was then replaced with 100 μL of medium containing the extract. At specific time intervals (24, 48, and 72 hours), the medium was aspirated, and 100 μL of ECM solution containing 10% (v / v) CCK-8 reagent was added to each well. After incubation for another hour, the absorbance was measured at 450 nm using a multi-microplate reader (MultiskanEX, Thermo Fisher Scientific). Each experiment was repeated three times, and the relative growth rate (RGR), i.e., the percentage of surviving cells compared to the control group cultured in normal ECM medium, was calculated.

[0040] The effect of the microsphere extract on cell viability was further evaluated using the live / dead staining method: a concentration of 5 × 10⁻⁶ was used. 4 HVSMCs at a concentration of 1 / mL were co-cultured with microsphere extract in 24-well plates, with normal ECM medium as a control. The medium was aspirated at specific time intervals (24, 48, and 72 hours), and the plates were washed with PBS. Fluorescent working solution containing Calcein-AM and PI was added, and the plates were stained at 37°C for 15 minutes. Cell viability and morphology were observed using a confocal laser scanning microscope (CLSM).

[0041] 1.7 Blood compatibility test Blood compatibility is a key indicator for evaluating medical devices that come into direct contact with blood. The blood compatibility of different PBVHx microspheres was assessed using a hemolysis test: PBVHx microspheres were equilibrated with 5 mL of physiological saline at 37°C for 1 hour; fresh blood from New Zealand rabbits was anticoagulated with glucose citrate and diluted with 4 mL of blood to 5 mL of physiological saline to prepare a test blood solution.

[0042] Add 100 μL of diluted blood solution to each microsphere sample; incubate at 37°C for 1 hour, then centrifuge to separate the supernatant; measure the absorbance of the supernatant at 540 nm using a UV-Vis spectrophotometer. Physiological saline was used as a negative control (NEG, hemolysis rate 0%), and distilled water as a positive control (POS, hemolysis rate 100%). Each experiment was repeated three times, and the hemolysis rate was calculated using the following formula:

[0043] In the formula, ODt is the absorbance of the experimental group, ODneg is the absorbance of the negative control group, and ODpos is the absorbance of the positive control group.

[0044] Fresh blood from New Zealand rabbits containing heparin anticoagulant was collected and centrifuged at 2000 rpm for 10 minutes to separate plasma and platelets. The separated platelets were diluted 50 times with physiological saline to prepare a platelet suspension. 10 mg of embolic microspheres (EMs) were mixed with 2 mL of platelet suspension and incubated at 37 °C for 1 hour, followed by washing twice with 40 mL of pure water. The number and morphology of platelets adhering to the surface of the embolic microspheres were observed using a scanning electron microscope.

[0045] 1.8 Rabbit ear occlusion experiment Male New Zealand white rabbits weighing 2.5 ± 0.5 kg (purchased from Changsha Tianqin Biotechnology Co., Ltd., China) were used to study the embolization effect of microspheres. All animal experiments were conducted in accordance with procedures approved by the Northwestern University Laboratory Animal Management and Ethics Committee. Rabbits were chosen as the animal model because their central auricular artery is well-visible and of suitable size for embolization studies.

[0046] Rabbits were anesthetized by intravenous injection of sodium pentobarbital (30 mg / kg) and fixed supine on the operating table. The fur on both ears was shaved with an electric razor, and the area was disinfected with iodine tincture. A skin incision was made proximal to the ear to expose the central auricular artery. A 22G catheter with an outer ring was inserted into the inner ear, and the central needle was then removed. A 20 mg / mL microsphere glycerol suspension was slowly injected into the central auricular artery from proximal to distal. The control group received an equal volume of glycerol without microspheres. After endovascular embolization of the ear vessels, the injection site was pressed for 15 minutes, and the catheter was simultaneously removed. Photographs of ear infarction were taken at predetermined time intervals (1, 4, 7, 14, and 21 days) to assess the embolization effect.

[0047] 1.9 Statistical Analysis One-way ANOVA was performed using GraphPad Prism 8 software (GraphPad Inc., USA). Data are expressed as mean ± standard deviation, and P < 0.05 was considered statistically significant. In the data, ns indicates P > 0.05, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0048] 2. Results and Discussion 2.1 Preparation and Characterization of PBVHx Microspheres Using a modified G1 / O / W biemulsion method, TP@OPMs and control groups pSMs, pOPMs, and TP@SMs were successfully prepared. Figure 1 As expected, the particle size of TP@OPMs was 154.31 ± 31.17 μm. Figure 2 b), the surface has a large number of openings ( Figure 2a). After processing with WEI FOSM, the internal structure of TP@OPMs also exhibits an interconnected porous network. Figure 2 a) This is a typical open-pore structure feature. Random analysis of 100 surface pores and internal pores revealed that their particle sizes were 31.06±14.83 μm and 31.31±13.09 μm, respectively.

[0049] Unloaded TP pOPMs have similar structures and particle sizes to TP@OPMs. Figure 2 (a, b) There was no significant difference in particle size distribution between the surface and internal pores. Figure 2 c, d). The control group TP@SMs, however, has a traditional sealed structure, showing significant differences from the two microspheres mentioned above: TP@SMs has a smooth surface with very few micropores, maintaining a complete spherical structure; the interior is primarily sealed, with only a small number of micropores present in the outermost layer. Figure 2 a).

[0050] Therefore, it can be seen that the surface pores and internal pores of the open-cell PBVHx microspheres (TP@OPMs and pOPMs) are significantly larger than those of the sealed PBVHx microspheres (TP@SMs and pSMs). Figure 2 c, d). Furthermore, due to different structural designs, the water absorption rate and porosity of TP@OPMs are 2860% and 79.69%, respectively. Figure 2 e, f), significantly higher than TP@SMs (water absorption rate 7.73%, porosity 12.63%). Water absorption rate reflects the ratio of water absorbed by the microspheres to their own mass. The high water absorption rate of TP@OPMs is mainly attributed to its three-dimensional porous structure. Higher porosity means larger internal space and higher specific surface area, which is beneficial for increasing the contact between the microspheres and blood, thereby improving the dissolution rate of the material and the release rate of the drug.

[0051] In the FTIR spectrum of PBVHx microspheres, 2966 cm⁻¹ -1 2880cm -1 1380cm -1 The characteristic peak at 2930 cm⁻¹ is attributed to the CH₃ vibration. -1 1458cm -1 1282cm -1 1230cm -1 The characteristic peak at 1726 cm⁻¹ is attributed to the CH₂ vibration. -1 The characteristic peak at 1726 cm⁻¹ corresponds to the C=O group. Since TP contains both C=O and -OH groups, TP@OPMs exhibits a peak at 1726 cm⁻¹. -1 The vibration intensity increased at 3446cm. -1 The presence of a relatively wide absorption band indicates that TP has been successfully loaded onto the microspheres. Figure 2 g).

[0052] 2.2 In vitro TP release and degradation of PBVHx microspheres like Figure 3 As shown in figure a, the encapsulation efficiency of TP in TP@OPMs was 79±0.32%, slightly lower than that in TP@SMs (92±0.41%). This difference may be due to the carbon dioxide and ammonia produced by the decomposition of ammonium bicarbonate, which promote the escape of the drug from the organic solution during stirring.

[0053] To investigate degradation behavior, PBVHx microspheres with different TP-loaded structures were immersed in PBS containing 10 mg / mL lipase and subjected to in vitro degradation studies in a 37°C constant-temperature shaker. The results are as follows: Figure 3 As shown in b, the weight retention rates of both TP@OPMs and TP@SMs decreased over 21 days. In the first 14 days, the degradation curves of the two microspheres were similar; by day 21, the weight retention rate of TP@OPMs was 93.37%, significantly lower than that of TP@SMs (94.55%).

[0054] The drug loading capacity of PHA microspheres is related to their structural characteristics and the physicochemical properties of the drugs. Compared with sealed microspheres, porous microspheres are more conducive to drug delivery and controlled release. Their high porosity and large specific surface area can promote drug diffusion, cell adhesion, migration and proliferation, and even promote the growth of new tissues. Du et al. successfully loaded vancomycin onto PLGA porous microspheres and studied the effect of porosity on drug release. Shi et al. investigated the morphology and internal structural differences of PLA microspheres and their effects on drug release behavior, and finally found that the release curve is usually determined by the porosity of the microspheres. As a hydrophobic drug, TP can be successfully loaded onto PHA, PLGA and other microspheres in large quantities. Li et al. successfully prepared TPL@mPLGA with a particle size of 195.5±7.5 nm. The microspheres had good stability and a drug loading of 2.94%. Wu et al. successfully loaded hydrophobic DOX and TP into PLGA hybrid nanoparticles through self-assembly. The average encapsulation efficiencies of DOX and TP were 75.5% and 58.3%, respectively.

[0055] Scanning electron microscopy analysis showed that as PBVHx slowly degraded, tiny micropores appeared on the surface of TP@SMs. Figure 3 (As shown by the red arrow in d). Although TP@SMs exhibit a degradation trend, their sealed spherical structure remains largely intact. In contrast, TP@OPMs with open pore structures show more significant surface erosion over time, with visible cracks appearing around the pores. Figure 3(As shown by the blue arrow in section d), this may have accelerated the degradation process. However, the overall spherical structure of TP@OPMs remained relatively intact and did not collapse. Due to their larger specific surface area and greater contact with the lipase solution, TP@OPMs experienced more significant degradation than TP@SMs. However, during the short-term degradation phase, the mass loss of both types of microspheres did not exceed 10% (as indicated by the blue arrow in section d). Figure 3 d).

[0056] The interconnected porous structure and large specific surface area of ​​TP@OPMs also contribute to their faster drug release rate. On day 4 of cumulative release, the cumulative release rate of TP@OPMs reached 31.17%, significantly higher than that of TP@SMs (24.90%). Figure 3 c).

[0057] 2.3 Cytotoxicity To assess the feasibility of PBVHx microspheres as a potential in vivo embolic agent, their biocompatibility needs to be evaluated. The microspheres were incubated in culture medium to obtain extracts at different incubation times. These extracts were then co-cultured with HVSMCs to study their cytotoxicity. Figure 4 a). For example Figure 4 As shown in c, the extracts obtained after microsphere incubation for 24, 48, and 72 hours had no significant effect on the viability of HVSMCs (p>0.05), and the cell viability remained above 89.21%.

[0058] To further validate the cytotoxicity results, a live / dead staining experiment was performed on HVSMCs co-cultured with the microsphere extract. After one day of co-culture with the extract, live cells (green staining) were evenly distributed, with only a small number of dead cells (red staining). Figure 4 c), which is consistent with the results of the CCK-8 experiment.

[0059] PHA is a natural biopolymer synthesized by microorganisms, renowned for its excellent biodegradability, biocompatibility, and adjustable degradation rate. The biodegradation products of PHA (3HB or 4HB monomers or oligomers) are less acidic and less irritating to humans. At the same molecular weight, PHA's degradation products are safer than other biodegradable polyester materials (such as PLA, PLGA, etc.).

[0060] PLGA and PLA play important roles in tissue engineering and drug delivery due to their unique biocompatibility and biodegradability. However, these polymers release lactic acid during degradation, which may have adverse effects on surrounding tissues. Lactic acid accumulation may lead to acidification of the tumor microenvironment, thereby promoting tumor invasion and metastasis, and affecting normal cell function and tissue health. For example, in lung cancer, long-term lactic acid exposure to non-small cell lung cancer cells increases histone lactation levels, significantly promoting tumor cell growth and proliferation; in addition, lactic acid can also promote the growth and proliferation of colorectal cancer by regulating the Wnt signaling pathway. Lactic acid accumulation may also suppress immune responses, including inhibiting the proliferation of immune effector cells, inducing immune cell dedifferentiation, leading to suppressed anti-tumor immune responses, activating potent negative regulators of adaptive immune cells, enabling tumors to evade immune surveillance, and further promoting cancer progression.

[0061] Therefore, although PLGA and PLA have broad application prospects in the medical field, their lactate release rate, release amount, and potential risks to surrounding tissues must be fully considered when designing and applying these materials. In contrast, PHA can achieve long-term drug release, is less irritating to tissues, and its degradation products are safe for humans and have no genotoxicity, making it very suitable for drug delivery and tissue engineering.

[0062] 2.4 In vitro antitumor effects The in vitro antitumor activity of pSMs, pOPMs, TP@OPMs, and TP@SMs against HepG2 cells was evaluated using the CCK-8 assay. Figure 5 As shown in Figure a, TP@OPMs were placed in Transwell chambers, allowing TP to be released from the microspheres and diffuse into HepG2 cells in the culture plate. Within the first 12 hours, the relative growth rates (RGR) of HepG2 cells treated with TP@OPMs and TP@SMs were 74.98% and 64.08%, respectively, indicating that both microspheres possessed significant antitumor activity, but the difference between the two groups was not statistically significant (p>0.05 or ns). Figure 5 (b) At 24 and 48 hours, the HepG2 cells treated with TP@OPMs showed the lowest relative growth rates (47.67% and 18.75%, respectively), indicating superior antitumor efficacy compared to the TP@SMs treatment group. Conversely, HepG2 cells treated with unloaded pSMs and pOPMs maintained relative growth rates above 95%, further confirming the excellent cell compatibility of blank PBVHx microspheres regardless of their structure.

[0063] Compared to other anticancer drugs (such as cisplatin, paclitaxel, and DOX), TP exhibits stronger inhibitory activity against hepatocellular carcinoma. TP inhibits the activation of the STAT3 signaling pathway by preventing STAT3 from binding to DNA, thereby reducing the expression of STAT3-regulated genes and inducing apoptosis. Furthermore, TP effectively inhibits NF-κB activity while activating p38α and ERK1 / 2, leading to decreased protein expression of the NF-κB subunits c-Rel and RelA, as well as a decrease in IκBα phosphorylation. Moreover, TP can block Dsh activation by inhibiting LRP6 phosphorylation, thereby inhibiting the Wnt / β-catenin signaling pathway. TP can also induce DNA demethylation, further inhibiting the Wnt signaling pathway and increasing cellular sensitivity to apoptosis.

[0064] Hydrogels, due to their excellent flowability and biodegradability, are often used as embolic agents to reduce the toxicity of hydrophilic drugs and maintain sustained anticancer effects. However, TP, as a typical hydrophobic drug, cannot be stably dispersed in hydrogels. To solve this problem, this invention uses a novel hydrophobic biopolyester PBVHx, achieving a high TP loading rate of 79% and enabling controlled drug release for up to 20 days, thereby reducing side effects.

[0065] To further verify the superior anti-cancer effect of TP@OPMs, the number of HepG2 cells surviving after co-culture was analyzed. Figure 5 As shown in c, after co-culturing with different microspheres for 48 hours, the number of residual cells showed the following trend: control group (Ctr) ≈ pSMs ≈ pOPMs > TP@SMs > TP@OPMs, which is consistent with the relative growth rate results.

[0066] 2.5 Blood compatibility and platelet adhesion TP@OPMs and control groups (pSMs, OPMs, TP@SMs) were co-cultured with blood for 1 hour. Physiological saline and purified water were used as negative controls (NEG) and positive controls (POS), respectively, to determine the hemolysis rate of the microspheres. Figure 6 As shown in a and 6b, none of the four types of PBVHx embolization microspheres (pSMs, OPMs, TP@SMs, and TP@OPMs) exhibited hemolysis, with hemolysis rates of 0.32%, 0.43%, 0.30%, and 0.31%, respectively. These hemolysis rates were significantly lower than the 100% in the positive control group and also lower than the internationally recognized standard of 5.00%.

[0067] Scanning electron microscopy revealed that some platelets adhered to the surfaces of TP@OPMs and pOPMs, with slightly more platelets adhering than to pSMs and TP@SMs. Figure 6 c). This may be because the open structure is more conducive to platelet adhesion, thereby accelerating thrombus formation and completing the embolization process.

[0068] Previous studies have confirmed the good blood compatibility of PHA through quantitative research on platelet adhesion and hemolysis rate assessment on the surface of PHA microspheres. Recent research also indicates that PHA materials have potential advantages in blood compatibility, particularly in resisting hemolysis and platelet adhesion. These properties make PHA an ideal material for medical devices, especially those that need to come into contact with blood.

[0069] 2.6 Study on ear vascular embolism To evaluate the embolic effects of TP@OPMs, pSMs, pOPMs, TP@SMs, and control glycerol and commercially available gelatin particles (cGPs), macroscopic observation was conducted to monitor changes in the morphology and color of rabbit ear vessels over 21 days. Figure 7 a). For example Figure 7 As shown in b, 21 days after glycerol injection into the central auricular artery (control group), no obvious color or morphological abnormalities appeared at the injection site, and no tissue necrosis occurred. This is because glycerol diffuses with the bloodstream and cannot block blood vessels in the ear, consistent with previous reports.

[0070] Conversely, injection of four types of PBVHx embolization microspheres into the auricular artery induced varying degrees of tissue necrosis. On day 7 post-embolization, the TP@OPMs, pOPMs, and TP@SMs treatment groups showed localized skin darkening and necrosis, while the pSMs treatment group and the control group did not exhibit necrosis. The necrosis rate showed the following trend: TP@OPMs > TP@SMs ≈ pOPMs > pSMs ≈ cGP ≈ control group (…). Figure 7 c).

[0071] On day 14, continuous blood flow occlusion led to skin blackening and necrosis in the pSMs treatment group. Prolonged ischemia can cause varying degrees of tissue necrosis. Simultaneously, the necrotic areas expanded in the TP@OPMs, TP@SMs, and pOPMs treatment groups, while the cGP treatment group only showed mild necrosis. The necrosis rate at this stage was ranked as follows: TP@OPMs > TP@SMs > pOPMs > pSMs > cGP ≈ control group. Notably, the necrosis rate of drug-loaded PBVHx embolization microspheres (TP@OPMs and TP@SMs) was almost twice that of pure PBVHx embolization microspheres (pOPMs and pSMs). Figure 7 c).

[0072] On day 21, tissue detachment occurred in all PBVHx embolization microsphere treatment groups, while skin necrosis began to appear distal to the ear vessels in the cGP treatment group. TP@OPMs showed the best embolization effect, with a necrosis rate of 50.70%. The necrosis rate at this stage was ranked as follows: TP@OPMs > TP@SMs > pOPMs ≈ pSMs > cGP > control group. Figure 7c). These results indicate that all PBVHx embolic microspheres prepared in this invention can effectively block blood vessels, with TP@OPMs exhibiting the most significant embolic effect.

[0073] 3. Summary Porous PBVHx microspheres loaded with triptolide (TP) (TP@OPMs) were prepared, and their anticancer and embolic activity were compared with those of sealed PBVHx microspheres loaded with TP (TP@SMs). TP@OPMs have a structure characterized by open surfaces and interconnected internal pores, resulting in higher water absorption (2860%) and porosity (79.69%), but slightly lower TP encapsulation efficiency (79±0.32%) compared to TP@SMs. They also achieved sustained TP release of 75.29% within 21 days. All unloaded PBVHx microspheres exhibited good biocompatibility, while TP@OPMs showed better inhibitory effects on HepG2 cells than TP@SMs, with a hemolysis rate of less than 5%. In in vivo central artery embolization experiments, TP@OPMs outperformed other microspheres and commercially available gelatin microspheres, achieving the best embolic effect with a necrosis rate of 50.70%, successfully inducing ischemic necrosis and tissue sloughing. These results demonstrate that TP@OPMs are not only an effective drug delivery system, but also have great potential in embolization applications, providing a promising strategy for the treatment of hepatocellular carcinoma and other malignancies.

[0074] Example 2 Based on the above Example 1, the application of the prepared TP@OPMs in the preparation of chemoembolization agents for the treatment of hepatocellular carcinoma is disclosed.

[0075] Example 3 Based on the above-described Example 1, the application of the prepared TP@OPMs in the preparation of vascular embolization devices for inducing tumor ischemic necrosis is disclosed.

[0076] Example 4 Based on the above Example 1, an embolic microsphere composition is disclosed, comprising TP@OPMs, a glycerol carrier and / or a dispersion medium; and the suspension concentration of TP@OPMs in glycerol is 20 mg / mL, while the glycerol carrier and / or dispersion medium are pharmaceutically acceptable substances and classified accordingly.

[0077] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A sustained-release embolic agent of tripterygium wilfordii with an open porous structure, characterized in that, The embolizing agent is a pore-filled PBVHx microsphere loaded with triptolide, denoted as TP@OPMs; The TP@OPMs have a porous structure with surface openings and interconnected internal pores.

2. The embolic agent of sustained-release tripterygium wilfordii with an open porous structure according to claim 1, characterized in that, The PBVHx contains 90 mol% 3-hydroxybutyric acid, 3 mol% 3-hydroxyvalerate and 7 mol% 3-hydroxyhexanoic acid, with a molecular weight of 46 kDa.

3. A preparation method for preparing TP@OPMs according to any one of claims 1-2, characterized in that, The steps are as follows: A. Dissolve PBVHx and TP together in dichloromethane; B. Mix the ammonium bicarbonate aqueous solution with the solution obtained in step A, and then homogenize and emulsify to form a G / O primary emulsion; C. Pour the G / O pre-emulsion into a polyvinyl alcohol aqueous solution, stir to evaporate dichloromethane, and form a G / O / W double emulsion; D. After centrifugation, washing, and freeze-drying, TP@OPMs were obtained.

4. The preparation method according to claim 3, characterized in that, The mass ratio of PBVHx to TP is 25:

1.

5. The preparation method according to claim 3, characterized in that, The ammonium bicarbonate aqueous solution has a mass-volume concentration of 20%.

6. The preparation method according to claim 3, characterized in that, The volume concentration of the polyvinyl alcohol aqueous solution is 1%.

7. The use of the TP@OPMs prepared according to claim 3 in the preparation of a chemoembolization formulation for the treatment of hepatocellular carcinoma.

8. The use of the TP@OPMs prepared according to claim 3 in the preparation of vascular embolization devices for inducing ischemic necrosis of tumors.

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